EZH2 Orchestrates Apicobasal Polarity and Neuroepithelial Cell Renewal

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EZH2 orchestrates apicobasal polarity and neuroepithelial cell renewal

Naiara Akizu & Marian A. Martínez-Balbás

To cite this article: Naiara Akizu & Marian A. Martínez-Balbás (2016) EZH2 orchestrates apicobasal polarity and neuroepithelial cell renewal, Neurogenesis, 3:1, e1250034, DOI: 10.1080/23262133.2016.1250034 To link to this article: http://dx.doi.org/10.1080/23262133.2016.1250034

Accepted author version posted online: 17 Nov 2016. Published online: 17 Nov 2016.

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Download by: [4.14.225.180] Date: 17 February 2017, At: 10:31 NEUROGENESIS 2016, VOL. 3, NO. 1, e1250034 (7 pages) http://dx.doi.org/10.1080/23262133.2016.1250034

COMMENTARY EZH2 orchestrates apicobasal polarity and neuroepithelial cell renewal

Naiara Akizu* and Marian A. Martınez-Balbas Department of Molecular Genomics, Instituto de Biologıa Molecular de Barcelona (IBMB), Consejo Superior de Investigaciones Cientıﬁcas (CSIC), Barcelona, Spain

ABSTRACT ARTICLE HISTORY During early stages of neural development, neuroepithelial cells translocate their nuclei along the Received 18 July 2016 apicobasal axis in a harmonized manner with the cell cycle. How cell cycle progression and Revised 12 October 2016 neuroepithelium polarity are coordinated remains unclear. It has been proposed that developmental Accepted 13 October 2016 cues, epigenetic mechanisms and cell cycle regulators must be linked in order to orchestrate these KEYWORDS processes. We have recently discovered that a master epigenetic factor, EZH2 is essential to coordinate WAF1/CIP apicobasal polarity; EZH2; these events. EZH2 directly represses the cell cycle regulator p21 in the chicken spinal cord. By gene silencing; histone doing so, EZH2 controls neural progenitor cell renewal and ﬁne-tunes Rho signaling pathway, which is methylation; neuroblast essential to maintain neuroepithelial structure. Our ﬁndings point to a new role of EZH2 during proliferation; neurogenesis development that could have potential implication in other areas as cancer.

The development of the nervous system is a highly orga- responsible for this activity are Enhancer of Zeste nized process where bipolar neuroepithelial cells play a Homolog’s 1 and 2 (EZH1/2)6 that belong to the Poly- key role. During neurogenesis these cells divide to expand comb Repressive Complex2 (PRC2). H3K27me3 is the neural progenitor pool and generate differentiated recognized by Polycomb proteins leading to transcrip- neural cells. Their cell cycle is coordinated with the apico- tional repression of many developmental regulators.7-9

basal interkinetic nuclear migration in such a way that This mark is removed by JMJD3 and UTX demethy- mitosis occurs in the apical membrane.1,2 It is believed lase activity.10-13 H3K27me3 has shown to play a role that external developmental cues, epigenetic mechanisms in hereditary transmission of chromatin states and and cell cycle regulators might be linked in order to coor- structure. Although it is still unclear how H3K27me3 dinate these processes. Although apicobasal polarity is levels are restored after several DNA replication known to influence self-renewal at early neurogenesis,3 rounds and mitosis, it has been proposed that the the exact molecular mechanisms underlying this coordi- unmodified newly synthetized histones incorporated nation program remain unclear. in the daughter strands get the H3K27me3 mark During the last decade, great efforts have been through the recruitment of PRC2 to the older made to elucidate the role of epigenetic regulators that H3K27me3. This mechanism also accounts for mito- govern different aspects of neural development. The sis, maintaining H3K27me3 at targets genes through epigenetic control, understood as the heritable the cell cycle14 and allows to preserve transcriptional changes in genome activity that do not involve alter- programs and cellular identity during development. ation of the DNA sequence, is mainly mediated by PRC2 controls proliferation of progenitors cells, in covalent modifications of histones and DNA.4 part by repressing of the Ink4A/Arf locus,15-17 and reg- Recently, histone methylation has received special ulates cellular differentiation.8,16,18,19 In addition to its attention as an essential regulator of gene expression. role as an epigenetic factor, EZH2 cooperates with dif- In particular, methylation of lysine 27 of histone H3 ferent signals in the cytoplasm to allow actin reorgani- (H3K27me3) has been found to be an important regu- zation.20 Although the function of EZH2 as a lator of embryonic development.5 The enzymes transcriptional repressor is well characterized, its role

CONTACT Marian A. Martınez-Balbas [email protected]; Naiara Akizu [email protected] Department of Molecular Genomics. Instituto de Biología Molecular de Barcelona (IBMB), Consejo Superior de Investigaciones Cientıﬁcas (CSIC), Barcelona Science Park. Helix Building, Room 02-19 C/ Baldiri Reixac 15-21. Barcelona 08028, Spain. *Present address: Dorris Neuroscience Center, Department of Molecular and Cellular Neuroscience, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA. © 2016 Taylor & Francis e1250034-2 N. AKIZU AND M. A. MARTINEZ-BALBAS during vertebrate development, and particularly in revealing a new mechanism that coordinates neuro- neurogenesis, is just emerging. blast proliferation and neuroepithelium (NE) structure.27 We observed that EZH2 is highly expressed in EZH2 in neurogenesis the ventricular zone of the chicken neural tube, where the neuroblasts reside, and dramatically decreased in Research efforts in the last decade have highlighted the the mantle zone, where the differentiated neurons relevance of EZH2 and Polycomb proteins in a wide accumulate. By in ovo electroporation of speciﬁc range of neurogenic processes. During the development shRNAs, we depleted EZH2 resulting in a small neural of the cerebral cortex, conditional deletion of Ezh2 at tube. The reduced size was due to the blocking of self- early embryonic stages induces premature neuronal dif- renewal of neuroepithelial cells and cell death by apo- ferentiation, which leads to a decreased number of neu- ptosis. Remarkably, the EZH2 knock down (K ) rons at birth.21 At later stages, EZH2 and Polycomb D embryos showed, in addition, a structurally disorga- proteins repress Ngn1 expression. By doing so Polycomb nized neural tube: the apical membrane was severely proteins regulate the timing of neurogenic to astrogenic disrupted along the luminal surface, and a subset of fate switch that occurs as the cortex develops.22 cells lost the apical junctions and invaded the neural In addition to its role in the cerebral cortex EZH2 is a tube lumen. Moreover, functional, ectopic lumens critical player in the development of the cerebellum. Ezh2 were observed in the EZH2 K neural tubes. is highly expressed in the cerebellar primordium as early D It is well established that Rho signaling pathway cou- as embryonic day E10.5 and regulates transcription of ples developmental signals downstream cytoskeletal rear- multiple genes involved in proliferation of progenitor rangements and cell proliferation.28 Interestingly, Rho cells and differentiation of cerebellar neuronal lineages. pathway is subjected to a strict spatiotemporal regulation Accordingly, deletion of Ezh2 in the dorsal neural tube, in the neural tube29 and it is repressed at early embryonic leads to underdevelopment of the cerebellum.23 But the neural tubes.30,31 These data led us to test whether EZH2 function of EZH2 in hindbrain development goes even could affect the NE structure by regulating Rho activity at further. The use of a sophisticated combination of genetic

the early stages of neurogenesis. Our results indicated tools has recently revealed a complex role of EZH2 in that the observed structural alterations were concomitant precerebellar neuron migration, mostly mediated by the with increased Rho activity in the EZH2 depleted transcriptional regulation of the guidance ligand-receptor embryos. These data reveal an unexpected new role of molecules in migrating cells and their environment. EZH2: in addition to allow the self-renewal and survival Either the deletion of Ezh2 from the migrating precere- of neural progenitor cells, EZH2 is required to maintain bellar neurons or from the surrounding neural tube cells apicobasal polarity and NE integrity. alters their final localization and connectivity.24 More recently, an essential role of EZH2 in adult WAF1/CIP1 neurogenesis has been reported. In hippocampal neu- EZH2-mediated p21 expression ral progenitor cell EZH2 regulates proliferation by coordinates apicobasal polarity and cell suppressing Pten expression and promoting the acti- proliferation vation of Akt-mTOR. As a consequence, conditional To gain insight into the role of EZH2 on progenitor deletion of Ezh2 results in defective hippocampal neu- self-renewal and NE apicobasal polarity at a molecular rogenesis and impaired spatial learning and mem- level, we analyzed the EZH2-mediated transcriptional ory.25 Furthermore, EZH2 is also required for fate profile by microarray experiments. We found that determination of adult neural progenitor cells. p21WAF1/CIP1, a well-known tumor suppressor gene, Although functional relevance in humans is still was highly up regulated in EZH2 K neural tubes. unknown, EZH2 expression was found in adult neural D Interestingly, restoration of p21WAF1/CIP1 levels in progenitor cells suggesting that EZH2 might be key EZH2 depleted embryos rescued not only proliferation for postnatal human neurogenesis as well.26 defects but also many of the NE structural alterations and partially restored the Rho activity levels. More- EZH2 in the spinal cord development over, we demonstrated that cytoplasmic p21WAF1/CIP1 Our recent report provides new insight into the EZH2 (but not the nuclear p21WAF1/CIP1) induced the forma- function at early neurogenesis in the spinal cord, tion of ectopic and functional lumens, similar but NEUROGENESIS e1250034-3 milder to those observed in EZH2 depleted neural tubes. Our data suggested that the increase on Rho activity mediated by EZH2-depletion was partially due to high levels of p21WAF1/CIP1. However, the par- tial rescue of Rho activity by p21WAF1/CIP1 depletion, and the milder NE structural defects by electroporation of cytoplasmic p21WAF1/CIP1 indicated that additional factors might contribute to Rho activity in EZH2 depleted spinal cord. Consistent with this, the expression arrays showed that some Rho family members, RhoBTB2, and ARHGAP10 were regulated by EZH2. Moreover, EZH2 non transcriptional effects need to be taken into account, since cytoplasmic EZH2 regulates actin polymerization by interacting with vav1, an activator of RhoA GTPase signaling.20 One of the unsolved questions in early neurogenesis is how neural tube structure and cell renewal are coordinated. Our results bring light to this problem. At Figure 1. A model for EZH2-mediated regulation of neurogenesis early stages of development, EZH2 keeps silenced at early stages of development in the spinal cord. EZH2 keeps silenced some Rho signaling components and the cell cycle regu- some Rho signaling components and the cell cycle reg- lator p21WAF1/CIP1 at the ventricular zone of the neural tube. In WAF1/CIP1 ulator p21 . By doing so, EZH2 allows the this way, EZH2 ensures the correct establishment of apicobasal establishment of apicobasal polarity that ensures polarity that allows the neuroepithelial cell self-renewal (left proper cell renewal (Fig. 1). Future investigations of panel). In EZH2 depleted embryos, an increased Rho activity due to low EZH2 activity and increased p21WAF1/CIP1 levels leads to a this model will provide additional data of the process structurally altered neural tube where the neuroblasts do not WAF1/CIP1 by which p21 regulates the Rho signaling proliferate (right panel). Red dots on the chromatin indicate pathway and the contribution of non transcriptional H3K27me3. Darker areas on the neural tube refer to differenti- role of EZH2 on Rho activity regulation as has been ated neurons. previously proposed.20 EZH2 overexpression is commonly found in adult and pediatric primary brain tumors as well.33 Implications for medicine in central nervous According to most of the reported studies, molecu- system lar mechanism underlying oncogenic activity of EZH2 EZH2 is well known by its role as an oncogene. More points toward its methyltransferase activity on H3K27 than a decade ago, the overexpression of EZH2 was and consequent target gene repression. Although associated with prostate cancer malignancy.32 Since p16INK4A/p19ARF locus was one of the first Polycomb then, multiple studies have shown that high levels of targets found misregulated in a mouse model of gli- EZH2 correlate with tumor cell proliferation, aggres- oma36 most recent transcriptional profiling studies sive behavior and poor prognosis in a wide range of have identified additional groups of EZH2 target genes cancers, including primary brain tumors.33-35 For involved in cell cycle regulation, stem cell differentia- instance, overexpression of EZH2 correlates with poor tion and cellular migration.33 However, just a handful prognosis of glioblastoma,35 one of the most aggres- of these genes, have been shown to act as effectors of sive primary brain tumors due to its rapid progression EZH2 oncogenic activity in brain tumors. Frequently, and invasiveness. EZH2 is indeed necessary for main- these effectors are also regulated by EZH2 in neural tenance and self-renewal of glioblastoma cancer stem stem cells during embryonic development. This is the cells, since its pharmacological inhibition or depletion case of BMPR1B, which progressive expression pro- with interference RNA impairs proliferation and motes astrocyte differentiation from neural stem cells tumor initiation capabilities of the glioblastoma cancer during development, but persistent repression by stem cells. Additional studies on medulloblastoma, EZH2 in glioblastoma stem cells contribute to tumor ependidoma and neuroblastoma have revealed that cell proliferation and maintenance.37 e1250034-4 N. AKIZU AND M. A. MARTINEZ-BALBAS

H3K27 methyltransferase activity of EZH2 is also prostate, breast and melanoma.45,46 Likewise, core- fundamental in the chicken embryo spinal cord. As pression of E-cadherin by Snail and EZH2 is also a key we observed, EZH2 guarantees neural stem cell self- step for migration of neural crest cells during develop- renewal and proliferation, in part, by repressing ment.47 In the ventricular zone of the developing p21WAF1/CIP1 expression. In addition to their onco- embryo however, loss of neuroepithelial structure is genic and tumor suppressor activity respectively, driven by EZH2 depletion and consequent induction EZH2 and p21WAF1/CIP1 have antagonistic roles on of Rho GTPase activity by cytoplasmic p21WAF1/CIP1 senescence.38 Cellular senescence is an irreversible as our work has revealed. Moreover, we also found growth arrest induced by cellular damage or stress additional Rho GTPase family members activated by conditions. Consistent with its role as a tumor sup- EZH2 KD that could contribute to the loss of neuroe- pressor mechanism, senescence in premalignant pithelial structure and invasive behavior of certain tumors avoid progression of tumorigenesis, and inac- cells in our model. Hence, our work suggests a counter tivation of senescence activating proteins, typically intuitive tumorigenic mechanism of EZH2 depletion. p16CDKN2A, is found in full-blown cancers.39-41 How- Indeed, homozygous or heterozygous deletions of ever, recent ﬁndings indicate that developmental cues EZH2 are common in malignant myeloid diseases;48-50 also activate senescence during embryogenesis con- and both activating and inactivating mutations of tributing to the development and morphogenesis of EZH2 are related to malignancy.51,52 Although inacti- multiple embryonic tissues.42,43 Interestingly, one of vating EZH2 mutations are not found in brain tumors the major players of developmental senescence is yet, lysine to methionine mutations of the residue 27 p21WAF1/CIP1. But does this have any relevance for the of the histone H3 tail, are recurrently found in several nervous system development and diseases? aggressive pediatric brain tumors, likely resembling Other than the presence of senescent cells that cor- consequences of EZH2 depletion.53 In addition, relates with p21WAF1/CIP1 expression,42 little is known knockout of EZH2 cells with Ink4a/Arf locus deletion about the role of senescence in the neural tube devel- do not inhibit cell proliferation and de-represses 26

opment. Preliminary data from our lab indicates that Olig2, an oncogenic driver of glial tumors. Moreover, p21WAF1/CIP1 is expressed in the mantle zone, where EZH2 positive tumors, including glioblastoma tumor we also observed postmitotic senescent neural cells. cells, develop resistance to pharmacological inhibition 54 Although senescence was not evaluated in EZH2 KD of EZH2, suggesting that persistent repression of neural tubes, through p21WAF1/CIP1 repression in the EZH2 can also contribute to malignancy. Thus, the ventricular zone, EZH2 might act blocking senescence. observation that EZH2 regulates the activity of Rho Indeed, several aspects of the phenotype we observe GTPase and the tumor suppressor p21WAF1/CIP1 knocking down EZH2 could result from the activation expression opens new avenues to fully understand the of senescence, for example, the non-cell autonomous role of EZH2 in cancer and metastasis. behavior of certain Tuj1 positive differentiated cells or Clinical relevance of EZH2, however, goes beyond migration of cells that invade the lumen. One of the its oncogenic activity. Recent work identified that high hallmarks of senescent cells is the senescence associ- levels of EZH2 and H3K27me3 are present in the cere- ated secretory phenotype (SASP) that allows signaling bellum of Ataxia Telangiectasia patients, a devastating from senescent cells to the external environment condition caused by ATM protein kinase deficiency. through the secretion of growth factors, cytokines/ Remarkably, reduction of EZH2 levels rescued Pur- chemiokines and extracellular remodeling factors. In kinje cell degeneration and behavioral defects of an some cases SASP has been implicated in epithelial Ataxia Telangiectasia mouse model, pointing toward a mesenchymal transition (EMT) and invasion of neigh- promising future therapeutic strategy for these boring cells.44 patients.55 EMT is one of the first steps that a tumor cell In summary, our work uncovers an intricate cross- undergoes to become metastatic. This process requires talk between factors that modify chromatin, cell cycle loss of epithelial polarity and cell adhesion, typically and cell polarity factors that fine-tune spinal cord triggered by repression of cell adhesion molecules like development. We have described a new role for EZH2 E-cadherin. The repression of E-cadherin by EZH2 is regulating cell renewal and neuroepithelial structure well established in a variety of tumors, including in the spinal cord. Underlying molecular mechanisms NEUROGENESIS e1250034-5 that our work revealed will hopefully provide new 2002; 298:1039-43; PMID:12351676; http://dx.doi.org/ opportunities to target context dependent EZH2 func- 10.1126/science.1076997 tion in different physiological and pathological [8] Boyer LA, Plath K, Zeitlinger J, Brambrink T, Medeiros LA, LeeTI,LevineSS,WernigM,TajonarA,RayMK,etal.Poly- conditions. comb complexes repress developmental regulators in murine embryonic stem cells. Nature 2006; 441:349-53; Abbreviations PMID:16625203; http://dx.doi.org/10.1038/nature04733 EZH1/2 Enhancer of Zeste Homolog’s 1 and 2 [9] Ringrose L, Paro R. Polycomb/Trithorax response ele- H3K27me3 Trimethylation of lysine 27 of histone ments and epigenetic memory of cell identity. Develop- H3 ment 2007; 134:223-32; PMID:17185323; http://dx.doi. org/10.1242/dev.02723 K Knock down D [10] Agger K, Cloos PA, Christensen J, Pasini D, Rose S, Rap- NE Neuroepithelium psilber J, Issaeva I, Canaani E, Salcini AE, Helin K. UTX PRC2 Polycomb Repressive Complex2 and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Nature 2007; Disclosure of potential conflicts of interest 449:731-4; PMID:17713478; http://dx.doi.org/10.1038/ nature06145 No potential conflicts of interest were disclosed. [11] De Santa F, Totaro MG, Prosperini E, Notarbartolo S, Testa G, Natoli G. The histone H3 lysine-27 demethylase Jmjd3 Acknowledgments links inflammation to inhibition of polycomb-mediated We want to thank Dr Xavier de la Cruz for corrections and for gene silencing. Cell 2007; 130:1083-94; PMID:17825402; critical reading of the manuscript. http://dx.doi.org/10.1016/j.cell.2007.08.019 [12] Lan F, Bayliss PE, Rinn JL, Whetstine JR, Wang JK, Chen Funding S, Iwase S, Alpatov R, Issaeva I, Canaani E, et al. A his- Our study was supported by grants BFU-2012-34261 to tone H3 lysine 27 demethylase regulates animal posterior MAMB from the Spanish Ministry of Education and Science, development. Nature 2007; 449:689-94; PMID:17851529; 090210 from Fundacio La Marato de TV3 and Fondation http://dx.doi.org/10.1038/nature06192 Jerome^ Lejeune to MAMB. [13] Lee MG, Villa R, Trojer P, Norman J, Yan KP, Reinberg

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